Structured Review

TaKaRa caspase 8
Fig. 6. Activities of <t>caspase-8</t> ( A ) and caspase-9 ( B ) in TRX-2 –/– /Trx-2 cells. Caspase-8 and -9 activities were determined using the fluorescent substrates IETD–AFC and LEVD–AMC at days 0, 3 and 5 after dox treatment. Etoposide was used as a positive control. ( C ) Cytochrome c release in the cytoplasm was observed by western blotting using anti-cytochrome c antibody in the cytoplasmic fraction of TRX-2 –/– /Trx-2 cells at days 0, 3, 5 and 7 after dox treatment. ( D ) Analysis of membrane potential by fluorescence-activated cell sorting (FACS) using DiCO 6 at days 0, 3 and 5 after dox treatment. CCCP was used as a positive control.
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1) Product Images from "Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis"

Article Title: Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis

Journal: The EMBO Journal

doi: 10.1093/emboj/21.7.1695

Fig. 6. Activities of caspase-8 ( A ) and caspase-9 ( B ) in TRX-2 –/– /Trx-2 cells. Caspase-8 and -9 activities were determined using the fluorescent substrates IETD–AFC and LEVD–AMC at days 0, 3 and 5 after dox treatment. Etoposide was used as a positive control. ( C ) Cytochrome c release in the cytoplasm was observed by western blotting using anti-cytochrome c antibody in the cytoplasmic fraction of TRX-2 –/– /Trx-2 cells at days 0, 3, 5 and 7 after dox treatment. ( D ) Analysis of membrane potential by fluorescence-activated cell sorting (FACS) using DiCO 6 at days 0, 3 and 5 after dox treatment. CCCP was used as a positive control.
Figure Legend Snippet: Fig. 6. Activities of caspase-8 ( A ) and caspase-9 ( B ) in TRX-2 –/– /Trx-2 cells. Caspase-8 and -9 activities were determined using the fluorescent substrates IETD–AFC and LEVD–AMC at days 0, 3 and 5 after dox treatment. Etoposide was used as a positive control. ( C ) Cytochrome c release in the cytoplasm was observed by western blotting using anti-cytochrome c antibody in the cytoplasmic fraction of TRX-2 –/– /Trx-2 cells at days 0, 3, 5 and 7 after dox treatment. ( D ) Analysis of membrane potential by fluorescence-activated cell sorting (FACS) using DiCO 6 at days 0, 3 and 5 after dox treatment. CCCP was used as a positive control.

Techniques Used: Positive Control, Western Blot, Fluorescence, FACS

2) Product Images from "Candida albicans Killing by RAW 264.7 Mouse Macrophage Cells: Effects of Candida Genotype, Infection Ratios, and Gamma Interferon Treatment †"

Article Title: Candida albicans Killing by RAW 264.7 Mouse Macrophage Cells: Effects of Candida Genotype, Infection Ratios, and Gamma Interferon Treatment †

Journal: Infection and Immunity

doi: 10.1128/IAI.70.11.6319-6329.2002

Caspase-8 (A) and caspase-9 (B) activities of RAW 264.7 macrophage cells in the presence of C. albicans strains. Macrophage cells incubated without Candida and those treated with UV correspond to negative and positive controls, respectively. Cell samples were harvested at the indicated times, and caspase-8 and -9 activities were determined as described in Materials and Methods.
Figure Legend Snippet: Caspase-8 (A) and caspase-9 (B) activities of RAW 264.7 macrophage cells in the presence of C. albicans strains. Macrophage cells incubated without Candida and those treated with UV correspond to negative and positive controls, respectively. Cell samples were harvested at the indicated times, and caspase-8 and -9 activities were determined as described in Materials and Methods.

Techniques Used: Incubation

3) Product Images from "Cordyceps militaris and mycelial fermentation induced apoptosis and autophagy of human glioblastoma cells"

Article Title: Cordyceps militaris and mycelial fermentation induced apoptosis and autophagy of human glioblastoma cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2012.172

Caspase activation in human glioblastoma cells. Cultured human glioblastoma GBM8401 ( a ) and U-87MG ( b ) cells were treated with CM or mycelial fermentation (MF) for 6 h at 2 mg/ml, followed by lysate collection, protein quantification, and subsequent luminometrical detection for activities of caspase-3, caspase-8, and caspase-9. Caspase activity readings were normalized by total protein. The representative data in arbitrary luminescent unit are shown as mean±S.D. from three independent experiments. * P
Figure Legend Snippet: Caspase activation in human glioblastoma cells. Cultured human glioblastoma GBM8401 ( a ) and U-87MG ( b ) cells were treated with CM or mycelial fermentation (MF) for 6 h at 2 mg/ml, followed by lysate collection, protein quantification, and subsequent luminometrical detection for activities of caspase-3, caspase-8, and caspase-9. Caspase activity readings were normalized by total protein. The representative data in arbitrary luminescent unit are shown as mean±S.D. from three independent experiments. * P

Techniques Used: Activation Assay, Cell Culture, Activity Assay

4) Product Images from "Expression and modulation of translocator protein and its partners by hypoxia reoxygenation or ischemia and reperfusion in porcine renal models"

Article Title: Expression and modulation of translocator protein and its partners by hypoxia reoxygenation or ischemia and reperfusion in porcine renal models

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.90422.2008

Effect of H 2 O 2 in the different experimental groups. Cell injury was measured by LDH ( A ) and MTT assay ( B ). Caspase 8 ( C ) and -9 ( D ) activities were determined and are presented, respectively. * P
Figure Legend Snippet: Effect of H 2 O 2 in the different experimental groups. Cell injury was measured by LDH ( A ) and MTT assay ( B ). Caspase 8 ( C ) and -9 ( D ) activities were determined and are presented, respectively. * P

Techniques Used: MTT Assay

5) Product Images from "Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal"

Article Title: Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg210

Fig. 4. HBx hyperactivates caspase-3 by TNF-α, and the activation is dependent on caspase-8. Caspase-3 activity in HepG2 ( A ) or Chang liver ( B ) cells and the effect of caspase-8 inhibitor on the caspase-8 ( C ) and caspase-3 ( D ) activity in HepG2 cells transfected with pEGFP, pEG-HBx and pEG-HBx-GFP with TNF-α treatment. At 10 h post-transfection, cells were treated with TNF-α for 40 h (A) or 18 h (B), and analyzed for caspase-3 activity. At 10 h post-transfection, HepG2 cells were treated with TNF-α and caspase-8 inhibitor (Ac-IETD-CHO, 1 µg/ml) for 40 h and analyzed for caspase-8 (C) and caspase-3 (D) activity. All results are the mean of three independent experiments, and the activities are presented relative to the control pEGFP vector.
Figure Legend Snippet: Fig. 4. HBx hyperactivates caspase-3 by TNF-α, and the activation is dependent on caspase-8. Caspase-3 activity in HepG2 ( A ) or Chang liver ( B ) cells and the effect of caspase-8 inhibitor on the caspase-8 ( C ) and caspase-3 ( D ) activity in HepG2 cells transfected with pEGFP, pEG-HBx and pEG-HBx-GFP with TNF-α treatment. At 10 h post-transfection, cells were treated with TNF-α for 40 h (A) or 18 h (B), and analyzed for caspase-3 activity. At 10 h post-transfection, HepG2 cells were treated with TNF-α and caspase-8 inhibitor (Ac-IETD-CHO, 1 µg/ml) for 40 h and analyzed for caspase-8 (C) and caspase-3 (D) activity. All results are the mean of three independent experiments, and the activities are presented relative to the control pEGFP vector.

Techniques Used: Activation Assay, Activity Assay, Transfection, Plasmid Preparation

Fig. 3. HBx hyperactivates caspase-8 through death signals. Caspase-8 activity in HepG2 ( A ) or Chang liver ( B ) cells transfected with 1 µg of pEGFP, pEG-HBx and pEG-HBx-GFP upon TNF-α treatment. At 10 h post-transfection, cells were treated with TNF-α for 40 h (A) or 18 h (B), and the caspase-8 activity was analyzed as described in Materials and methods. ( C ) Caspase-8 activity of Chang cells transfected with pEGFP, pEG-HBx and pEG-HBx-GFP following anti-Fas antibody treatment. At 20 h post-transfection, cells were treated with anti-Fas antibody and cycloheximide (20 µg/ml) for 6 h. Cells were washed and treated with Fab antibody for cross-linking for 30 min before harvest, and caspase-8 activity was analyzed. All results are the mean of three independent experiments. ( D ) Cleavage of procaspase-8 to the active subunit (p18) in HepG2 cells transfected with the indicated plasmids following TNF-α treatment. HepG2 cells were transfected with pEG-HBx (lanes 1–3), pEG-HBx-GFP (lanes 4–6) and pEGFP (lanes 7–9). At 10 h post-transfection, cells were treated with TNF-α (0, 0.1 and 10.0 ng/ml, respectively) for 40 h. Total cell lysates were normalized for protein concentration, separated by 12% SDS–PAGE and analyzed by western blotting. The caspase activity is shown relative to the control vector pEGFP transfection.
Figure Legend Snippet: Fig. 3. HBx hyperactivates caspase-8 through death signals. Caspase-8 activity in HepG2 ( A ) or Chang liver ( B ) cells transfected with 1 µg of pEGFP, pEG-HBx and pEG-HBx-GFP upon TNF-α treatment. At 10 h post-transfection, cells were treated with TNF-α for 40 h (A) or 18 h (B), and the caspase-8 activity was analyzed as described in Materials and methods. ( C ) Caspase-8 activity of Chang cells transfected with pEGFP, pEG-HBx and pEG-HBx-GFP following anti-Fas antibody treatment. At 20 h post-transfection, cells were treated with anti-Fas antibody and cycloheximide (20 µg/ml) for 6 h. Cells were washed and treated with Fab antibody for cross-linking for 30 min before harvest, and caspase-8 activity was analyzed. All results are the mean of three independent experiments. ( D ) Cleavage of procaspase-8 to the active subunit (p18) in HepG2 cells transfected with the indicated plasmids following TNF-α treatment. HepG2 cells were transfected with pEG-HBx (lanes 1–3), pEG-HBx-GFP (lanes 4–6) and pEGFP (lanes 7–9). At 10 h post-transfection, cells were treated with TNF-α (0, 0.1 and 10.0 ng/ml, respectively) for 40 h. Total cell lysates were normalized for protein concentration, separated by 12% SDS–PAGE and analyzed by western blotting. The caspase activity is shown relative to the control vector pEGFP transfection.

Techniques Used: Activity Assay, Transfection, Protein Concentration, SDS Page, Western Blot, Plasmid Preparation

Fig. 10. A proposed model for the enhanced susceptibility to the death signal due to HBx. HBx forms a complex with c-FLIP L and c-FLIP S in the cytoplasm, abrogating their apoptosis-inhibitory function. The recruitment of c-FLIP to DISC is blocked by the interaction with HBx and, consequently, caspase-8 and caspase-3 are hyperactivated by death signals even below the threshold concentration that renders the cells susceptible to apoptosis.
Figure Legend Snippet: Fig. 10. A proposed model for the enhanced susceptibility to the death signal due to HBx. HBx forms a complex with c-FLIP L and c-FLIP S in the cytoplasm, abrogating their apoptosis-inhibitory function. The recruitment of c-FLIP to DISC is blocked by the interaction with HBx and, consequently, caspase-8 and caspase-3 are hyperactivated by death signals even below the threshold concentration that renders the cells susceptible to apoptosis.

Techniques Used: Concentration Assay

Fig. 8. Overexpression of c-FLIP rescues the cells from HBx-mediated apoptosis through a MAPK-independent pathway. ( A ) HepG2 cells were co-transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with dimethylsulfoxide (DMSO; filled bar) or PD98059 (10 µM, open bar). After 1 h incubation, cells were treated with TNF-α (1 ng/ml) for 47 h and stained with annexin V–PE and 7-AAD. The viability was quantified by the annexin V and 7-AAD double-negative cells after GFP gating by flow cytometry. ( B ) HepG2 cells were co-transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with TNF-α (1 ng/ml) for 48 h and the viability analyzed without gating by flow cytometry. ( C ) Effect of c-FLIP, caspase-8 and several inhibitors on the viability of cells transfected with pEG-HBx-GFP. HepG2 cells were co-transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with DMSO (filled bar), cycloheximide (10 µg/ml, open bar) and Ac-IETD-CHO (2 µM, dotted bar). After 1 h incubation, cells were treated with TNF-α (1 ng/ml) for 47 h and the viability was quantified. The graph represents the ratio of viability relative to the control GFP vector. All the results are the mean of three independent experiments. ( D ) Cell viability of HepG2 cells transfected with pEG-HBx and pcDNA (left), pEG-HBx and pcDNA (middle), or pEG-HBx and pcDNA3-cFLIP-L (right). At 12 h post-transfection, cells were treated with DMSO or Ac-IETD-CHO (2 µM). After 1 h incubation, cells were treated with TNF-α (1 ng/ml) for 47 h. The photographs were taken at 60 h after co-transfection by phase-contrast microscopy.
Figure Legend Snippet: Fig. 8. Overexpression of c-FLIP rescues the cells from HBx-mediated apoptosis through a MAPK-independent pathway. ( A ) HepG2 cells were co-transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with dimethylsulfoxide (DMSO; filled bar) or PD98059 (10 µM, open bar). After 1 h incubation, cells were treated with TNF-α (1 ng/ml) for 47 h and stained with annexin V–PE and 7-AAD. The viability was quantified by the annexin V and 7-AAD double-negative cells after GFP gating by flow cytometry. ( B ) HepG2 cells were co-transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with TNF-α (1 ng/ml) for 48 h and the viability analyzed without gating by flow cytometry. ( C ) Effect of c-FLIP, caspase-8 and several inhibitors on the viability of cells transfected with pEG-HBx-GFP. HepG2 cells were co-transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with DMSO (filled bar), cycloheximide (10 µg/ml, open bar) and Ac-IETD-CHO (2 µM, dotted bar). After 1 h incubation, cells were treated with TNF-α (1 ng/ml) for 47 h and the viability was quantified. The graph represents the ratio of viability relative to the control GFP vector. All the results are the mean of three independent experiments. ( D ) Cell viability of HepG2 cells transfected with pEG-HBx and pcDNA (left), pEG-HBx and pcDNA (middle), or pEG-HBx and pcDNA3-cFLIP-L (right). At 12 h post-transfection, cells were treated with DMSO or Ac-IETD-CHO (2 µM). After 1 h incubation, cells were treated with TNF-α (1 ng/ml) for 47 h. The photographs were taken at 60 h after co-transfection by phase-contrast microscopy.

Techniques Used: Over Expression, Transfection, Incubation, Staining, Flow Cytometry, Cytometry, Plasmid Preparation, Cotransfection, Microscopy

6) Product Images from "The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation"

Article Title: The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.051378298

) with the pCMV control plasmid (cont.), the DCC expression plasmid pDCC-CMV-S (DCC), or the DCC expression plasmid and the netrin-1-encoding plasmid pGNET1myc (DCC + net.) in the presence of the p35 expression construct pBabe-p35 ( A ), or of the dominant negative caspase-3, -8, or -9 expression construct ( B . ( C ) Mock (cont.) or DCC (DCC) transfected 293T cells were incubated or not with 20 μM Zvad-fmk (Zvad), IETD-fmk (IETD), DEVD-fmk (DEVD), or LEHD-fmk (LEHD) 24 h after the beginning of the transfection. Cell death was then analyzed by using trypan blue (as in A and B ) 24 h later. ( D ) Caspase-9 −/− cells were transfected with a mock plasmid (cont.), DCC-expressing plasmid (DCC), or the caspase-8-expressing plasmid pcDNA-casp.8 (casp.8). ( E and F ) IMR32 cells were cultured for 48 h in the presence of culture medium issued of 293-EBNA-netrin-1 cells producing extracellular netrin-1. After this conditioned growth, cells were either further cultured with netrin-1-containing medium (+ net.) or with medium devoid of netrin-1 (− net.). In the latter case, cells were incubated or not with 20 μM LEHD-fmk (+LEHD). Cell death was then measured either by trypan blue staining at the indicated time following netrin-1 withdrawal ( E ) or by TUNEL reactivity after 24 h of netrin-1 withdrawal ( F ).
Figure Legend Snippet: ) with the pCMV control plasmid (cont.), the DCC expression plasmid pDCC-CMV-S (DCC), or the DCC expression plasmid and the netrin-1-encoding plasmid pGNET1myc (DCC + net.) in the presence of the p35 expression construct pBabe-p35 ( A ), or of the dominant negative caspase-3, -8, or -9 expression construct ( B . ( C ) Mock (cont.) or DCC (DCC) transfected 293T cells were incubated or not with 20 μM Zvad-fmk (Zvad), IETD-fmk (IETD), DEVD-fmk (DEVD), or LEHD-fmk (LEHD) 24 h after the beginning of the transfection. Cell death was then analyzed by using trypan blue (as in A and B ) 24 h later. ( D ) Caspase-9 −/− cells were transfected with a mock plasmid (cont.), DCC-expressing plasmid (DCC), or the caspase-8-expressing plasmid pcDNA-casp.8 (casp.8). ( E and F ) IMR32 cells were cultured for 48 h in the presence of culture medium issued of 293-EBNA-netrin-1 cells producing extracellular netrin-1. After this conditioned growth, cells were either further cultured with netrin-1-containing medium (+ net.) or with medium devoid of netrin-1 (− net.). In the latter case, cells were incubated or not with 20 μM LEHD-fmk (+LEHD). Cell death was then measured either by trypan blue staining at the indicated time following netrin-1 withdrawal ( E ) or by TUNEL reactivity after 24 h of netrin-1 withdrawal ( F ).

Techniques Used: Plasmid Preparation, Droplet Countercurrent Chromatography, Expressing, Construct, Dominant Negative Mutation, Transfection, Incubation, Cell Culture, Staining, TUNEL Assay

Related Articles

Colorimetric Assay:

Article Title: Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal
Article Snippet: .. The activities of caspase-8 and caspase-3 were measured by the caspase ApoAlert colorimetric assay kit (Clontech) using a synthetic peptidyl substrate IETD-pNA and DEVD-pNA, respectively. .. Briefly, 1–2 × 106 cells were treated with TNF-α or anti-Fas antibody, and lysed in cell lysis buffer on ice.

Amplification:

Article Title: Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation
Article Snippet: .. The primer sequences were as follows: BAX forward primer 5′-AGACACCTGAGCTGACCT TGGA-3′and reverse primer 5′-GAGACACTCGCTCAGCTTCTTG-3′; BCL2 forward primer 5′-TGCCACCTGTGGTCCATCT-3′and reverse primer 5′-GTGCAGCTGACTGGACATCTCT-3′; caspase 9 forward primer 5′-TCACGGCTTTGATGGAGATG-3′and reverse primer 5′-GAGGATGACCACCACAAAGCA-3′; caspase 7 forward primer 5′-ACAGCAACTCGGCCTGCTT-3′and reverse primer 5′-TTCCCGT AAATCAGGTCCTCTTC-3′; caspase 8 forward primer 5′-AAGGCAA TCTGTCTTTCCTGAAA-3′and reverse primer 5′-TCGGTTGCAGTC TAGGAAGTTG-3′; TGF-beta 1 forward primer 5′-TGGAGCAACAT GTGGAACTC-3′and reverse primer 5′-GTCAGCAGCCGGTTAC CA-3′; and HPRT forward primer 5′-GAAAGACTTGCTCGAGATGT CATG-3′and reverse primer 5′-CACACAGAGGGCCACAATGT-3′. cDNA generated was amplified in Sybr Advantage qPCR Premix (Clontech, Mountain View, CA), consisting of a full-length Taq™ poly-merase with a hot-start Taq antibody and SYBR Green I, by using an ABI 7500 Fast Real Time PCR System (PE Applied Biosystems). .. A sample volume of 20 μl was used for all assays and contained a 1× final concentration of SYBR Advantage Premix, 250 n M gene-specific primers, and 2 μl cDNA template.

Fluorescence:

Article Title: Candida albicans Killing by RAW 264.7 Mouse Macrophage Cells: Effects of Candida Genotype, Infection Ratios, and Gamma Interferon Treatment †
Article Snippet: .. Caspase-8 and caspase-9 activities were assayed with ApoAlert caspase fluorescence assay kits (Clontech) in accordance with the manufacturer's instructions. ..

Article Title: Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis
Article Snippet: .. The activities of caspase-8 and caspase-9 were measured using IETD–AFC (7-amino-4-trifluoromethyl coumarin) or LEHD–AMC (7-amino-4-methyl coumarin) as substrate (caspase-8 and -9 fluorescent assay kit; Clontech), and detected as the emission shift of AFC or AMC. ..

Article Title: Expression and modulation of translocator protein and its partners by hypoxia reoxygenation or ischemia and reperfusion in porcine renal models
Article Snippet: .. Caspase 8 and caspase 9 activity assays were performed with ApoAlert caspase fluorescent assay kits (Clontech, Palo Alto, CA). .. ATP levels were quantified using a luminometry ATP Assay kit (Thermo Labsystems, Franklin, MA) according to the manufacturer's instructions.

Clone Assay:

Article Title: A New Caspase-8 Isoform Caspase-8s Increased Sensitivity to Apoptosis in Jurkat Cells
Article Snippet: .. The PCR products of the CDS of caspase-8, caspase-8s and FADD were cloned into the pMD18-T vector (Takara, Japan) and sequenced by ABI PRISM 377-96 genetic analyzer (Applied Biosystems, USA). ..

Caspase-3 Activity Assay:

Article Title: The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation
Article Snippet: .. Caspase-8 and caspase-3 activity were measured by using the ApoAlert caspase-8, ApoAlert caspase-3 assays from CLONTECH and the caspase-3 activity assay from Boehringer Mannheim. ..

Caspase Assay:

Article Title: Cordyceps militaris and mycelial fermentation induced apoptosis and autophagy of human glioblastoma cells
Article Snippet: .. Caspase activity assay The lysates from the cells with 6 h of treatment were subjected to assessment of caspase-3, caspase-8, and caspase-9 activities by an ApoAlert caspase assay kit (Clontech, Mountain View, CA, USA). .. The caspase activity was luminometrically measured by Luminoskan Ascent microplate reader (Thermo Scientific, Vienna, VA, USA) and readings were normalized by total protein.

SYBR Green Assay:

Article Title: Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation
Article Snippet: .. The primer sequences were as follows: BAX forward primer 5′-AGACACCTGAGCTGACCT TGGA-3′and reverse primer 5′-GAGACACTCGCTCAGCTTCTTG-3′; BCL2 forward primer 5′-TGCCACCTGTGGTCCATCT-3′and reverse primer 5′-GTGCAGCTGACTGGACATCTCT-3′; caspase 9 forward primer 5′-TCACGGCTTTGATGGAGATG-3′and reverse primer 5′-GAGGATGACCACCACAAAGCA-3′; caspase 7 forward primer 5′-ACAGCAACTCGGCCTGCTT-3′and reverse primer 5′-TTCCCGT AAATCAGGTCCTCTTC-3′; caspase 8 forward primer 5′-AAGGCAA TCTGTCTTTCCTGAAA-3′and reverse primer 5′-TCGGTTGCAGTC TAGGAAGTTG-3′; TGF-beta 1 forward primer 5′-TGGAGCAACAT GTGGAACTC-3′and reverse primer 5′-GTCAGCAGCCGGTTAC CA-3′; and HPRT forward primer 5′-GAAAGACTTGCTCGAGATGT CATG-3′and reverse primer 5′-CACACAGAGGGCCACAATGT-3′. cDNA generated was amplified in Sybr Advantage qPCR Premix (Clontech, Mountain View, CA), consisting of a full-length Taq™ poly-merase with a hot-start Taq antibody and SYBR Green I, by using an ABI 7500 Fast Real Time PCR System (PE Applied Biosystems). .. A sample volume of 20 μl was used for all assays and contained a 1× final concentration of SYBR Advantage Premix, 250 n M gene-specific primers, and 2 μl cDNA template.

Generated:

Article Title: Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation
Article Snippet: .. The primer sequences were as follows: BAX forward primer 5′-AGACACCTGAGCTGACCT TGGA-3′and reverse primer 5′-GAGACACTCGCTCAGCTTCTTG-3′; BCL2 forward primer 5′-TGCCACCTGTGGTCCATCT-3′and reverse primer 5′-GTGCAGCTGACTGGACATCTCT-3′; caspase 9 forward primer 5′-TCACGGCTTTGATGGAGATG-3′and reverse primer 5′-GAGGATGACCACCACAAAGCA-3′; caspase 7 forward primer 5′-ACAGCAACTCGGCCTGCTT-3′and reverse primer 5′-TTCCCGT AAATCAGGTCCTCTTC-3′; caspase 8 forward primer 5′-AAGGCAA TCTGTCTTTCCTGAAA-3′and reverse primer 5′-TCGGTTGCAGTC TAGGAAGTTG-3′; TGF-beta 1 forward primer 5′-TGGAGCAACAT GTGGAACTC-3′and reverse primer 5′-GTCAGCAGCCGGTTAC CA-3′; and HPRT forward primer 5′-GAAAGACTTGCTCGAGATGT CATG-3′and reverse primer 5′-CACACAGAGGGCCACAATGT-3′. cDNA generated was amplified in Sybr Advantage qPCR Premix (Clontech, Mountain View, CA), consisting of a full-length Taq™ poly-merase with a hot-start Taq antibody and SYBR Green I, by using an ABI 7500 Fast Real Time PCR System (PE Applied Biosystems). .. A sample volume of 20 μl was used for all assays and contained a 1× final concentration of SYBR Advantage Premix, 250 n M gene-specific primers, and 2 μl cDNA template.

Activity Assay:

Article Title: The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation
Article Snippet: .. Caspase-8 and caspase-3 activity were measured by using the ApoAlert caspase-8, ApoAlert caspase-3 assays from CLONTECH and the caspase-3 activity assay from Boehringer Mannheim. ..

Article Title: Expression and modulation of translocator protein and its partners by hypoxia reoxygenation or ischemia and reperfusion in porcine renal models
Article Snippet: .. Caspase 8 and caspase 9 activity assays were performed with ApoAlert caspase fluorescent assay kits (Clontech, Palo Alto, CA). .. ATP levels were quantified using a luminometry ATP Assay kit (Thermo Labsystems, Franklin, MA) according to the manufacturer's instructions.

Caspase Activity Assay:

Article Title: Cordyceps militaris and mycelial fermentation induced apoptosis and autophagy of human glioblastoma cells
Article Snippet: .. Caspase activity assay The lysates from the cells with 6 h of treatment were subjected to assessment of caspase-3, caspase-8, and caspase-9 activities by an ApoAlert caspase assay kit (Clontech, Mountain View, CA, USA). .. The caspase activity was luminometrically measured by Luminoskan Ascent microplate reader (Thermo Scientific, Vienna, VA, USA) and readings were normalized by total protein.

Polymerase Chain Reaction:

Article Title: A New Caspase-8 Isoform Caspase-8s Increased Sensitivity to Apoptosis in Jurkat Cells
Article Snippet: .. The PCR products of the CDS of caspase-8, caspase-8s and FADD were cloned into the pMD18-T vector (Takara, Japan) and sequenced by ABI PRISM 377-96 genetic analyzer (Applied Biosystems, USA). ..

Real-time Polymerase Chain Reaction:

Article Title: Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation
Article Snippet: .. The primer sequences were as follows: BAX forward primer 5′-AGACACCTGAGCTGACCT TGGA-3′and reverse primer 5′-GAGACACTCGCTCAGCTTCTTG-3′; BCL2 forward primer 5′-TGCCACCTGTGGTCCATCT-3′and reverse primer 5′-GTGCAGCTGACTGGACATCTCT-3′; caspase 9 forward primer 5′-TCACGGCTTTGATGGAGATG-3′and reverse primer 5′-GAGGATGACCACCACAAAGCA-3′; caspase 7 forward primer 5′-ACAGCAACTCGGCCTGCTT-3′and reverse primer 5′-TTCCCGT AAATCAGGTCCTCTTC-3′; caspase 8 forward primer 5′-AAGGCAA TCTGTCTTTCCTGAAA-3′and reverse primer 5′-TCGGTTGCAGTC TAGGAAGTTG-3′; TGF-beta 1 forward primer 5′-TGGAGCAACAT GTGGAACTC-3′and reverse primer 5′-GTCAGCAGCCGGTTAC CA-3′; and HPRT forward primer 5′-GAAAGACTTGCTCGAGATGT CATG-3′and reverse primer 5′-CACACAGAGGGCCACAATGT-3′. cDNA generated was amplified in Sybr Advantage qPCR Premix (Clontech, Mountain View, CA), consisting of a full-length Taq™ poly-merase with a hot-start Taq antibody and SYBR Green I, by using an ABI 7500 Fast Real Time PCR System (PE Applied Biosystems). .. A sample volume of 20 μl was used for all assays and contained a 1× final concentration of SYBR Advantage Premix, 250 n M gene-specific primers, and 2 μl cDNA template.

Plasmid Preparation:

Article Title: A New Caspase-8 Isoform Caspase-8s Increased Sensitivity to Apoptosis in Jurkat Cells
Article Snippet: .. The PCR products of the CDS of caspase-8, caspase-8s and FADD were cloned into the pMD18-T vector (Takara, Japan) and sequenced by ABI PRISM 377-96 genetic analyzer (Applied Biosystems, USA). ..

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    TaKaRa caspase 8 like ietdase
    Betanodavirus GGNNV infection induced activation of <t>caspase-8-like</t> proteases in SB cells. Cell lysates were harvested at 24 and 48 h PI and assayed for <t>IETDase</t> activity using caspase-8 colorimetric substrate IETD-pNA. Simultaneously, Mock-infected SB cells were used as a negative control. Values shown are means from duplicate experiments.
    Caspase 8 Like Ietdase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa injury assessment apoptosis caspase 8 activity
    Effects of lung ECM treatment on <t>apoptosis</t> and oxidative tissue damage in rats exposed to intermittent or continuous hyperoxia compared to saline treatment in hyperoxia-exposed and untreated normoxic control animals: <t>caspase-8</t> activity (A), 8-OHdG (B), protein carbonyl (C) and 8-isoprostane (D). Mean ± SD. p
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    Apoptosis induction by DMC in GBM 8401 cells via <t>caspase</t> 3, 8, and 9 activations. DMC activated pro-caspase-3 degradation in GBM 8401 cells. The cells were treated with DMC (0, 12.5, 25, and 50 μ M), and then (a) Western blot analysis was performed for pro- and cleaved- caspase-3. (b) Quantification of band intensities by Li-COR near-infrared imaging system. All data were reported as the means (±SEM) of at least three separate experiments. Statistical analysis used the t -test, with the significant differences determined at the level of * P
    Caspase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa caspase8
    Detection of apoptotic markers in cells of the testes after irradiation and TJ107 treatment. Spermatogenic cell apoptosis was assessed at day 120 in each group. Group A a , Group B b , Group C c and Group D d histological detection showed TUNEL staining in the testicular sections. Erratic round-shaped black areas in the seminiferous tubules indicate TUNEL-positive cells. Bar = 100 μm. e Expression of Fas , Fas ligand ( FasL ), and <t>caspase8</t> mRNA in mouse testes of each group at 120 days. Calculation of Relative mRNA intensity was done and the expression in the control group for each point was set to 1. The data are presented as mean ± standard deviation ( n = 10). Asterisks indicate p
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    Image Search Results


    Betanodavirus GGNNV infection induced activation of caspase-8-like proteases in SB cells. Cell lysates were harvested at 24 and 48 h PI and assayed for IETDase activity using caspase-8 colorimetric substrate IETD-pNA. Simultaneously, Mock-infected SB cells were used as a negative control. Values shown are means from duplicate experiments.

    Journal: Virology

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer

    doi: 10.1016/S0042-6822(02)00098-3

    Figure Lengend Snippet: Betanodavirus GGNNV infection induced activation of caspase-8-like proteases in SB cells. Cell lysates were harvested at 24 and 48 h PI and assayed for IETDase activity using caspase-8 colorimetric substrate IETD-pNA. Simultaneously, Mock-infected SB cells were used as a negative control. Values shown are means from duplicate experiments.

    Article Snippet: Colorimetric assay of caspases activity Colorimetric assay of caspase-8-like (IETDase) and caspase-3-like (DEVDase) proteolytic activity was performed using an ApoAlert Caspase-8 and Caspase-3 Colorimetric Assay Kit (Clontech Laboratories, Palo Alto, CA, USA).

    Techniques: Infection, Activation Assay, Activity Assay, Negative Control

    Effects of lung ECM treatment on apoptosis and oxidative tissue damage in rats exposed to intermittent or continuous hyperoxia compared to saline treatment in hyperoxia-exposed and untreated normoxic control animals: caspase-8 activity (A), 8-OHdG (B), protein carbonyl (C) and 8-isoprostane (D). Mean ± SD. p

    Journal: PLoS ONE

    Article Title: Lung protection by inhalation of exogenous solubilized extracellular matrix

    doi: 10.1371/journal.pone.0171165

    Figure Lengend Snippet: Effects of lung ECM treatment on apoptosis and oxidative tissue damage in rats exposed to intermittent or continuous hyperoxia compared to saline treatment in hyperoxia-exposed and untreated normoxic control animals: caspase-8 activity (A), 8-OHdG (B), protein carbonyl (C) and 8-isoprostane (D). Mean ± SD. p

    Article Snippet: Injury assessment Apoptosis: Caspase-8 activity was measured by colorimetric assay (ApoAlert™, Clontech, Mountain View, CA).

    Techniques: Activity Assay

    Apoptosis induction by DMC in GBM 8401 cells via caspase 3, 8, and 9 activations. DMC activated pro-caspase-3 degradation in GBM 8401 cells. The cells were treated with DMC (0, 12.5, 25, and 50 μ M), and then (a) Western blot analysis was performed for pro- and cleaved- caspase-3. (b) Quantification of band intensities by Li-COR near-infrared imaging system. All data were reported as the means (±SEM) of at least three separate experiments. Statistical analysis used the t -test, with the significant differences determined at the level of * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Demethoxycurcumin Retards Cell Growth and Induces Apoptosis in Human Brain Malignant Glioma GBM 8401 Cells

    doi: 10.1155/2012/396573

    Figure Lengend Snippet: Apoptosis induction by DMC in GBM 8401 cells via caspase 3, 8, and 9 activations. DMC activated pro-caspase-3 degradation in GBM 8401 cells. The cells were treated with DMC (0, 12.5, 25, and 50 μ M), and then (a) Western blot analysis was performed for pro- and cleaved- caspase-3. (b) Quantification of band intensities by Li-COR near-infrared imaging system. All data were reported as the means (±SEM) of at least three separate experiments. Statistical analysis used the t -test, with the significant differences determined at the level of * P

    Article Snippet: Caspase Activity Assay The caspase (2, 3, 8, and 9) activity was assessed by the ApoAlert Caspase assay plates (Clontech, USA).

    Techniques: Western Blot, Imaging

    Detection of apoptotic markers in cells of the testes after irradiation and TJ107 treatment. Spermatogenic cell apoptosis was assessed at day 120 in each group. Group A a , Group B b , Group C c and Group D d histological detection showed TUNEL staining in the testicular sections. Erratic round-shaped black areas in the seminiferous tubules indicate TUNEL-positive cells. Bar = 100 μm. e Expression of Fas , Fas ligand ( FasL ), and caspase8 mRNA in mouse testes of each group at 120 days. Calculation of Relative mRNA intensity was done and the expression in the control group for each point was set to 1. The data are presented as mean ± standard deviation ( n = 10). Asterisks indicate p

    Journal: BMC Complementary and Alternative Medicine

    Article Title: The effectiveness of traditional Japanese medicine Goshajinkigan in irradiation-induced aspermatogenesis in mice

    doi: 10.1186/s12906-019-2786-z

    Figure Lengend Snippet: Detection of apoptotic markers in cells of the testes after irradiation and TJ107 treatment. Spermatogenic cell apoptosis was assessed at day 120 in each group. Group A a , Group B b , Group C c and Group D d histological detection showed TUNEL staining in the testicular sections. Erratic round-shaped black areas in the seminiferous tubules indicate TUNEL-positive cells. Bar = 100 μm. e Expression of Fas , Fas ligand ( FasL ), and caspase8 mRNA in mouse testes of each group at 120 days. Calculation of Relative mRNA intensity was done and the expression in the control group for each point was set to 1. The data are presented as mean ± standard deviation ( n = 10). Asterisks indicate p

    Article Snippet: Subsequently, 3 ng of cDNA was amplified by 40 cycles of polymerase chain reaction (PCR) to measure Ki67 , Fas , FasL , caspase8 , claudin3 , claudin11 , occludin , ZO1 , ZO2 , and GAPDH , real-time fluorescence-monitored PCR was performed with the Thermal Cycler Dice Real-time System TP800 (TaKaRa) using TaqMan PCR Master Mix Reagents 2× and TaqMan Gene Expression Assays of primers 20×, according to the manufacturer’s instructions.

    Techniques: Irradiation, TUNEL Assay, Staining, Expressing, Standard Deviation